Botulinum Toxin A Upregulates Rac1, Cdc42, and RhoA Gene Expression in a Dose-Dependent Manner: In Vivo and in Vitro Study.

UNLABELLED: Angiogenesis is the development of new capillaries from existing blood vessels and is a prerequisite for the wound-healing process. Many lines of scientific evidences have shown that complicated roles of small guanosine triphosphatases (GTPases) (ras-related C3 botulinum toxin substrate 1 [Rac1], cell division control protein 42 [Cdc42], and ras homolog gene family, member A [RhoA]) in regulation of signal transduction pathways exist to transmit distinct cellular effects on the modulation of actin cytoskeleton remodeling such as cell cycle progression, cell survival, and cell motility. In addition, these small GTPases activate mitogen-activated protein kinase kinase kinases (MAP3Ks) leading to activated mitogen-activated protein kinase kinases (MAPKK), mitogen-activated protein kinase (MAPK), and various transcription factors such as vascular endothelial growth factor with involvement of MAPK signaling pathways.In this study, the authors hypothesized that botulinum toxin A increases angiogenesis via the expression of small GTPases in vivo and in vitro studies.In vivo experiment, 24 Sprague-Dawley rats were randomly divided into 2 groups: a control group and a botulinum toxin A group. Five days prior to superiorly based transverse rectus abdominis myocutaneous flap elevation, the botulinum toxin A (BoTA) group was pretreated with BoTA, while the control group was pretreated with normal saline. quantitative real-time polymerase chain reaction was performed to evaluate the expression of Rac1, RhoA, and Cdc42.The angiogenic effects of botulinum toxin A on human dermal fibroblasts were measured in vitro experiment. To understand the mechanism of botulinum toxin A on small GTPases production of fibroblasts, Rac1, Cdc42, and RhoA were measured using qRT-PCR.The relative messenger ribonucleic acid expression of Rac1, RhoA, and Cdc42 was significantly higher in the BoTA group than in the control group, in every zone and pedicle muscle, on postoperative days 1, 3, and 5. Levels of these molecules increased significantly in human dermal fibroblasts grown in the presence of BoTA compared with control group over 5 IU.Our in vivo and in vitro studies suggest that administration of BoTA upregulates the expression of RhoA, Rac1, and Cdc42 in a dose-dependent manner. MAPK signaling pathway might be involved in BoTA-induced angiogenesis mechanism. LEVEL OF EVIDENCE: N/A.

Effect of omega-3 polyunsaturated fatty acids on the cytoskeleton: an open-label intervention study.

BACKGROUND: Omega-3 polyunsaturated fatty acids (n-3 PUFAs) show beneficial effects on cardiovascular health and cognitive functions, but the underlying molecular mechanisms are not completely understood. Because of the fact that cytoskeleton dynamics affect almost every cellular process, the regulation of cytoskeletal dynamics could be a new pathway by which n-3 PUFAs exert their effects on cellular level. METHODS: A 12-week open-label intervention study with 12 healthy men was conducted to determine the effects of 2.7 g/d n-3 PUFA on changes in mRNA expression of cytoskeleton-associated genes by quantitative real-time PCR in whole blood. Furthermore, the actin content in red blood cells was analyzed by immunofluorescence imaging. RESULTS: N-3 PUFA supplementation resulted in a significant down-regulation of cytoskeleton-associated genes, in particular three GTPases (RAC1, RHOA, CDC42), three kinases (ROCK1, PAK2, LIMK), two Wiskott-Aldrich syndrome proteins (WASL, WASF2) as well as actin related protein 2/3 complex (ARPC2, ARPC3) and cofilin (CFL1). Variability in F-actin content between subjects was high; reduced actin content was only reduced within group evaluation. CONCLUSIONS: Reduced cytoskeleton-associated gene expression after n-3 PUFA supplementation suggests that regulation of cytoskeleton dynamics might be an additional way by which n-3 PUFAs exert their cellular effects. Concerning F-actin, this analysis did not reveal unmistakable results impeding a generalized conclusion.

A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42-mediated GSK-3ß/ß-catenin Signaling.

Kidney fibrosis is a common fate of chronic kidney diseases (CKDs), eventually leading to renal dysfunction. Yet, no effective treatment for this pathological process has been achieved. During the bioassay-guided chemical investigation of the medicinal plant Wikstroemia chamaedaphne, a daphne diterpenoid, daphnepedunin A (DA), is characterized as a promising anti-renal fibrotic lead. DA shows significant anti-kidney fibrosis effects in cultured renal fibroblasts and unilateral ureteral obstructed mice, being more potent than the clinical trial drug pirfenidone. Leveraging the thermal proteome profiling strategy, cell division cycle 42 (Cdc42) is identified as the direct target of DA. Mechanistically, DA targets to reduce Cdc42 activity and down-regulates its downstream phospho-protein kinase Cζ(p-PKCζ)/phospho-glycogen synthase kinase-3ß (p-GSK-3ß), thereby promoting ß-catenin Ser33/37/Thr41 phosphorylation and ubiquitin-dependent proteolysis to block classical pro-fibrotic ß-catenin signaling. These findings suggest that Cdc42 is a promising therapeutic target for kidney fibrosis, and highlight DA as a potent Cdc42 inhibitor for combating CKDs.

Verbascoside represses malignant phenotypes of esophageal squamous cell carcinoma cells by inhibiting CDC42 via the HMGB1/RAGE axis.

BACKGROUND: As an aggressive human malignancy, esophageal squamous cell carcinoma (ESCC) is prevalent globally, especially in China. Verbascoside (VE) exerts anti-cancer effects in several human cancers. This work was to investigate the effects of VE on ESCC cells. METHODS: Esophageal squamous cell carcinoma cell proliferation, apoptosis, migration, and invasion were assessed by CCK-8, TUNEL, and Transwell assays. Gene and protein levels were detected by RT-qPCR and western blotting. CDC42 activity was evaluated by G-lisa assay. RESULTS: Verbascoside significantly inhibited cell proliferation, migration, and invasion and induced cell apoptosis in ESCC cells. Furthermore, it was found that VE markedly inhibited HMGB1 and RAGE expression in a dose-dependent manner. Besides, HMGB1/RAGE upregulation partially reversed the anti-cancer effects of VE on ESCC cells. VE repressed HMGB1/RAGE-induced CDC42 activation in ESCC cells. In addition, ML141-mediated CDC42 inactivation further enhanced the effect of VE on ESCC cell proliferation, apoptosis, migration, and invasion. CONCLUSIONS: Our findings indicated that VE has significant anti-tumor potential in ESCC by suppressing HMGB1/RAGE-dependent CDC42 activation.

NSAIDs inhibit alpha V beta 3 integrin-mediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis.

Cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism, is overexpressed in many cancers. Inhibition of COX-2 by nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancer development in humans and suppresses tumor growth in animal models. The anti-cancer effect of NSAIDs seems to involve suppression of tumor angiogenesis, but the underlying mechanism is not completely understood. Integrin alpha V beta 3 is an adhesion receptor critically involved in mediating tumor angiogenesis. Here we show that inhibition of endothelial-cell COX-2 by NSAIDs suppresses alpha V beta 3-dependent activation of the small GTPases Cdc42 and Rac, resulting in inhibition of endothelial-cell spreading and migration in vitro and suppression of fibroblast growth factor-2-induced angiogenesis in vivo. These results establish a novel functional link between COX-2, integrin alpha V beta 3 and Cdc42-/Rac-dependent endothelial-cell migration. Moreover, they provide a rationale to the understanding of the anti-angiogenic activity of NSAIDs.

RPEL-family rhoGAPs link Rac/Cdc42 GTP loading to G-actin availability.

RPEL proteins, which contain the G-actin-binding RPEL motif, coordinate cytoskeletal processes with actin dynamics. We show that the ArhGAP12- and ArhGAP32-family GTPase-activating proteins (GAPs) are RPEL proteins. We determine the structure of the ArhGAP12/G-actin complex, and show that G-actin contacts the RPEL motif and GAP domain sequences. G-actin inhibits ArhGAP12 GAP activity, and this requires the G-actin contacts identified in the structure. In B16 melanoma cells, ArhGAP12 suppresses basal Rac and Cdc42 activity, F-actin assembly, invadopodia formation and experimental metastasis. In this setting, ArhGAP12 mutants defective for G-actin binding exhibit more effective downregulation of Rac GTP loading following HGF stimulation and enhanced inhibition of Rac-dependent processes, including invadopodia formation. Potentiation or disruption of the G-actin/ArhGAP12 interaction, by treatment with the actin-binding drugs latrunculin B or cytochalasin D, has corresponding effects on Rac GTP loading. The interaction of G-actin with RPEL-family rhoGAPs thus provides a negative feedback loop that couples Rac activity to actin dynamics.

Functional analysis of alpha5beta1 integrin and lipid rafts in invasion of epithelial cells by Porphyromonas gingivalis using fluorescent beads coated with bacterial membrane vesicles.

Porphyromonas gingivalis, a periodontal pathogen, was previously suggested to exploit alpha5beta1 integrin and lipid rafts to invade host cells. However, it is unknown if the functional roles of these host components are distinct from one another during bacterial invasion. In the present study, we analyzed the mechanisms underlying P. gingivalis invasion, using fluorescent beads coated with bacterial membrane vesicles (MV beads). Cholesterol depletion reagents including methyl-beta-cyclodextrin (MbetaCD) drastically inhibited the entry of MV beads into epithelial cells, while they were less effective on bead adhesion to the cells. Bead entry was also abolished in CHO cells deficient in sphingolipids, components of lipid rafts, whereas adhesion was negligibly influenced. Following MbetaCD treatment, downstream events leading to actin polymerization were abolished; however, alpha5beta1 integrin was recruited to beads attached to the cell surface. Dominant-negative Rho GTPase Rac1 abolished cellular engulfment of the beads, whereas dominant-negative Cdc42 did not. Following cellular interaction with the beads, Rac1 was found to be translocated to the lipid rafts fraction, which was inhibited by MbetaCD. These results suggest that alpha5beta1 integrin, independent of lipid rafts, promotes P. gingivalis adhesion to epithelial cells, while the subsequent uptake process requires lipid raft components for actin organization, with Rho GTPase Rac1.

NGF-dependent formation of ruffles in PC12D cells required a different pathway from that for neurite outgrowth.

Two signaling pathways, phosphoinositide 3-kinase (PI-3k)/Akt and Ras/MAPK, are major effectors triggered by nerve growth factor (NGF). Rac1, Cdc42 and GSK-3beta are reported to be targets of PI-3k in the signal transduction for neurite outgrowth. Immediately after NGF was added, broad ruffles were observed temporarily around the periphery of PC12 cells prior to neurite growth. As PC12D cells are characterized by a very rapid extension of neurites in response to various agents, the signaling pathways described above were studied in relation to the NGF-induced formation of ruffles and outgrowth of neurites. Wortmannin, an Akt inhibitor (V), and GSK-3beta inhibitor (SB425286) suppressed the neurite growth in NGF-treated cells, but not in dbcAMP-treated cells. The outgrowth of neurites induced by NGF but not by dbcAMP was inhibited with the expression of mutant Ras. But upon the expression of dominant-negative Rac1, cells often extended protrusions, incomplete neurites, lacking F-actin. Intact neurites were observed in cells with dominant-negative Cdc42. These results suggest that NGF-dependent neurite outgrowth occurs via a mechanism involving activation of the Ras/PI-3K/Akt/GSK-3beta pathway, while dbcAMP-dependent neurite growth might be induced in a distinct manner. However, inhibitors for GSK-3beta and PI-3k (wortmannin) did not suppress the NGF-dependent formation of ruffles. In addition, the formation of ruffles was not inhibited by the expression of mutant Ras. On the other hand, it was suppressed by the expression of dominant-negative Rac1 or Cdc42. These results suggest that the NGF-induced ruffling requires activation of Rac1 and Cdc42, but does not require Ras, PI-3k, Akt and GSK-3beta. Taken together, the NGF-dependent formation of ruffles might not require Ras/PI-3k/Akt/GSK-3beta, but these pathways might contribute to the formation of intact neurites due to combined actions including Rac1.

Actin can reorganize into podosomes in aortic endothelial cells, a process controlled by Cdc42 and RhoA.

Members of the Rho GTPase family play a central role in the orchestration of cytoskeletal rearrangements, which are of prime importance in endothelial cell physiology. To explore their role in this specialized cell type, we used the bacterial toxin cytotoxic necrotizing factor 1 (CNF1) as a Rho GTPase activator. Punctate filamentous actin structures appeared along the ventral plasma membrane of endothelial cells and were identified as the core of podosomes by the distinctive vinculin ring around the F-actin. Rho, Rac, and Cdc42 were all identified as targets of CNF1, but only a constitutively active mutant of Cdc42 could substitute for CNF1 in podosome induction. Accordingly, organization of F-actin in these structures was highly dependent on the main Cdc42 cytoskeletal effector N-Wiskott-Aldrich syndrome protein. Other components of the actin machinery such as Arp2/3 and for the first time WIP also colocalized at these sites. Like CNF1 treatment, sustained Cdc42 activity induced a time-dependent F-actin-vinculin reorganization, prevented cytokinesis, and downregulated Rho activity. Finally, podosomes were also detected on endothelial cells explanted from patients undergoing cardiac surgery. These data provide the first description of podosomes in endothelial cells. The identification of such specialized structures opens up a new field of investigation in terms of endothelium pathophysiology.

Activation of cdc42, rac, PAK, and rho-kinase in response to hepatocyte growth factor differentially regulates epithelial cell colony spreading and dissociation.

Hepatocyte growth factor (HGF), the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial-mesenchymal transition and dispersal of epithelial cells, processes that play crucial roles in tumor development, invasion, and metastasis. Little is known about the Met-dependent proximal signals that regulate these events. We show that HGF stimulation of epithelial cells leads to activation of the Rho GTPases, Cdc42 and Rac, concomitant with the formation of filopodia and lamellipodia. Notably, HGF-dependent activation of Rac but not Cdc42 is dependent on phosphatidylinositol 3-kinase. Moreover, HGF-induced lamellipodia formation and cell spreading require phosphatidylinositol 3-kinase and are inhibited by dominant negative Cdc42 or Rac. HGF induces activation of the Cdc42/Rac-regulated p21-activated kinase (PAK) and c-Jun N-terminal kinase, and translocation of Rac, PAK, and Rho-dependent Rho-kinase to membrane ruffles. Use of dominant negative and activated mutants reveals an essential role for PAK but not Rho-kinase in HGF-induced epithelial cell spreading, whereas Rho-kinase activity is required for the formation of focal adhesions and stress fibers in response to HGF. We conclude that PAK and Rho-kinase play opposing roles in epithelial-mesenchymal transition induced by HGF, and provide new insight regarding the role of Cdc42 in these events.

Major pathogenic effects of anti-MuSK antibodies in myasthenia gravis.

MG with anti-MuSK antibodies (MuSK+) is often characterized with muscle atrophy and excellent response to plasma exchanges. To elucidate some MuSK+ MG features, we analyzed the functional effects of anti-MuSK Abs in human TE 671 muscle cells. We found that some MuSK+ sera induced a striking inhibition of proliferation, accompanied by: 1) cell cycle arrest, 2) atrogin-1 overexpression, 3) AChR subunits, rapsyn, Rho A and cdc42 downregulation. These effects correlated to disease severity and to anti-MuSK Abs titer and vanished following PE. Altogether, these results indicate that anti-MuSK Abs could be pathogenic by contributing to the muscle atrophy in MuSK+ MG patients.

Endothelin suppresses cell migration via the JNK signaling pathway in a manner dependent upon Src kinase, Rac1, and Cdc42.

Cell migration is a complex phenomenon that is stimulated by chemoattractive factors such as chemokines, a family of ligands for G protein-coupled receptors (GPCRs). In contrast, factors that suppress cell migration, and the mechanism of their action, remain largely unknown. In this study, we show that endothelin, a GPCR ligand, inhibits cell motility in a manner dependent upon signaling through the c-Jun N-terminal kinase (JNK) pathway. We further demonstrate that this effect is dependent upon Src kinase and small GTPases Rac1 and Cdc42. These findings provide new insight into GPCR-mediated regulation of cell migration.

Targeting Cdc42 in cancer.

INTRODUCTION: The Rho GTPases are a family of proteins that control fundamental cellular processes in response to extracellular stimuli and internal programs. Rho GTPases function as molecular switches in which the GTP-bound proteins are active and GDP-bound proteins are inactive. This article will focus on one Rho family member, Cdc42, which is overexpressed in a number of human cancers, and which might provide new therapeutic targets in malignancies. AREAS COVERED: In this article, the key regulators and effectors of Cdc42 and their molecular alterations are described. The complex interactions between the signaling cascades regulated by Cdc42 are also analyzed. EXPERT OPINION: While mutations in Cdc42 have not been reported in human cancer, aberrant expression of Cdc42 has been reported in a variety of tumor types and in some instances has been correlated with poor prognosis. Recently, it has been shown that Cdc42 activation by oncogenic Ras is crucial for Ras-mediated tumorigenesis, suggesting that targeting Cdc42 or its effectors might be useful in tumors harboring activating Ras mutations.

Phenylboronic acid is a more potent inhibitor than boric acid of key signaling networks involved in cancer cell migration.

Previous studies from our lab have shown that both boric (BA) and phenylboronic- acid (PBA) inhibit the migration of prostate cancer cell lines, as well as non-tumorigenic prostate cells. Our results indicate that PBA is more potent than BA in targeting metastatic and proliferative properties of cancer cells. Here we focus on the impact of BA and PBA on Rho family of GTP-binding proteins and their downstream targets. Treatment with 1mM PBA and BA decreases activities of RhoA, Rac1, and Cdc42 in DU-145 metastatic prostate cancer cells, but not in normal RWPE-1 prostate cells. Furthermore, ROCKII activity and phosphorylation of myosin light chain kinase decrease as a result of either PBA or BA treatment in DU-145 cells, suggesting these compounds target actomyosin-based contractility.

TNFalpha induced oxidative stress dependent Akt signaling affects actin cytoskeletal organization in glioma cells.

Inflammation which is an indispensable participant in tumor progression is intricately linked with redox modulation. The pro-inflammatory cytokine Tumor Necrosis Factor (TNFalpha) elevates reactive oxygen species (ROS) in glioblastoma multiforme (GBM). As both TNFalpha and oxidative stress independently play role in regulating cytoskeletal organization and cell survival pathways we investigated whether TNFalpha mediated oxidative stress regulates responses that offer survival advantages to glioblastoma cells. Treatment with TNFalpha elevated Akt phosphorylation in glioma cells. Increased in Akt phosphorylation was concurrent with the decrease in ROS scavenger SOD-1 levels. TNFalpha mediated increase in Akt phosphorylation was dependent on oxidative stress as Akt phosphorylation was abrogated in the presence of ROS inhibitor and elevated in cells transfected with SOD-1 siRNA. TNFalpha altered actin cytoskeletal organization and increased Cdc42 levels. This increase in Cdc42 was concomitant with its increased interaction with scaffold protein IQGAP-1. Also, we report for the first time a ROS dependent interaction between pAkt and IQGAP-1 in TNFalpha treated cells. Importantly, Akt inhibition not only reversed TNFalpha mediated changes in actin cytoskeletal organization but also abrogated anchorage independent growth. Together, these results suggest that TNFalpha induced oxidative stress affects Akt activation to regulate actin organization and growth of glioma cells.

Rotenone-induced toxicity is mediated by Rho-GTPases in hippocampal neurons.

In this study, we have examined the effects of rotenone in primary cultures of hippocampal and dopaminergic neurons in order to obtain insights into the possible mechanisms underlying the neurotoxic effects of this pesticide. The results obtained indicate that a 48-h exposure to rotenone (0.1 microM) produces a complete and selective suppression of axon formation. This effect was dose dependent, not accompanied by changes in microtubule organization, and reversible after washout of the agrochemical from the tissue culture medium. Interestingly, pull-down assays revealed that rotenone decreases Cdc42 and Rac activities, whereas increasing that of Rho. In accordance with this, treatment of neuronal cultures with cytochalasin D, an actin-depolymerizing drug, or with the Rho-kinase inhibitor Y27632, or overexpression of Tiam1, a guanosine nucleotide exchange factor for Rac, reverts the inhibitory effect of rotenone on axon formation. Taken together, our data suggest that at least some of the neurotoxic effects of rotenone are associated with an inhibition of actin dynamics through modifications of Rho-GTPase activity.

Chronic ethanol treatment impairs Rac and Cdc42 activation in rat hepatocytes.

BACKGROUND: The effects of chronic ethanol feeding on rat hepatocytes have been shown to include impaired cell-extracellular matrix (ECM) adhesion events, such as decreased attachment and spreading as well as increased integrin-actin cytoskeleton association. These results, observed previously by this laboratory, are highly suggestive of impaired actin cytoskeleton reorganization, an event mediated by differential activation of the Rho family GTPases Rac, Cdc42, and RhoA. Therefore, the purpose of this study was to examine the effects of chronic ethanol administration on these GTPases. METHODS: Male Wistar rats were pair-fed 4 to 5 weeks with a liquid diet containing either ethanol (as 36% of total calories) or isocaloric carbohydrate. Hepatocytes were isolated and plated on collagen IV up to 24 hours. At specific times, the hepatocytes were lysed and these lysates were analyzed for RhoA, Cdc42, and Rac activation. RESULTS: In freshly isolated hepatocytes from ethanol-fed rats, the GTP-bound (active) forms of Rac and Cdc42 were significantly decreased compared with pair-fed control rats, while the GTP-bound form of RhoA was not significantly altered. These ethanol-induced impairments in Rac and Cdc42 activation persisted even after plating the hepatocytes on collagen IV. Additionally, chronic ethanol treatment did not directly affect GTP binding of Cdc42 and Rac, as incorporation of GTPgammaS was not affected. CONCLUSIONS: Chronic ethanol administration selectively impairs Rac and Cdc42 activation in rat hepatocytes. As activation of these 2 GTPases is crucial for efficient cell attachment and spreading on ECM substrates, the results from this study suggest that the ethanol-induced impairments in Rac and Cdc42 activation are responsible for the impaired hepatocyte-ECM adhesion events observed previously by our laboratory. Furthermore, these results raise the intriguing possibility that these GTPases are involved in other ethanol-induced functional impairments, such as protein trafficking and receptor-mediated endocytosis.

Molecular mechanisms contributing to dendritic spine alterations in the prefrontal cortex of subjects with schizophrenia.

Postmortem studies have revealed reduced densities of dendritic spines in the dorsal lateral prefrontal cortex (DLPFC) of subjects with schizophrenia. However, the molecular mechanisms that might contribute to these alterations are unknown. Recent studies of the intracellular signals that regulate spine dynamics have identified members of the RhoGTPase family (e.g., Cdc42, Rac1, RhoA) as critical regulators of spine structure. In addition, Duo and drebrin are spine-specific proteins that are critical for spine maintenance and spine formation, respectively. In order to determine whether the mRNA expression levels of Cdc42, Rac1, RhoA, Duo or drebrin are altered in schizophrenia, tissue sections containing DLPFC area 9 from 15 matched pairs of subjects with schizophrenia and control subjects were processed for in situ hybridization. Expression levels of these mRNAs were also correlated with DLPFC spine density in a subset of the same subjects. In order to assess the potential influence of antipsychotic medications on the expression of these mRNAs, similar studies were conducted in monkeys chronically exposed to haloperidol or olanzapine. The expression of each of these mRNAs was lower in the gray matter of the subjects with schizophrenia compared to the control subjects, although only the reductions in Cdc42 and Duo remained significant after corrections for multiple comparisons. In addition, spine density was strongly correlated with the expression levels of both Duo (r=0.73, P=0.007) and Cdc42 (r=0.71, P=0.009) mRNAs. In contrast, the expression levels of Cdc42 and Duo mRNAs were not altered in monkeys chronically exposed to antipsychotic medications. In conclusion, reduced expression of Cdc42 and Duo mRNAs may represent molecular mechanisms that contribute to the decreased density of dendritic spines in the DLPFC of subjects with schizophrenia.

Resveratrol induces FasL-related apoptosis through Cdc42 activation of ASK1/JNK-dependent signaling pathway in human leukemia HL-60 cells.

Trans-resveratrol, a phytoalexin found at high levels in grapes and in grape products such as red wine, has been shown to prevent carcinogenesis or antitumor growth in murine models. Here we dissect the detailed signaling pathway involved in resveratrol-induced apoptosis. Our data showed that treatment with resveratrol-induced activation of apoptosis signal-regulating kinase 1, a mitogen-activated protein kinase kinase kinase, in turn, activated the downstream kinases c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, but not extracellular signal-regulated kinase. Transfection with a dominant-negative c-Jun N-terminal kinase expression vector reduced FasL expression and DNA fragmentation induced by resveratrol. However, inhibition of p38 mitogen-activated protein kinase activity by treatment with SB203580 (p38 mitogen-activated protein kinase specific inhibitor) or expression of mutant p38 mitogen-activated protein kinase expression vector did not alter the apoptosis and FasL expression in response to resveratrol. Furthermore, genetic inhibition of apoptosis signal-regulating kinase 1 signaling inhibited not only the activation of c-Jun N-terminal kinase, but also the expression of FasL and apoptosis. Similarly, over-expression of wild-type apoptosis signal-regulating kinase 1 strengthened the resveratrol-induced c-Jun N-terminal kinase activation, FasL expression and subsequent apoptosis. These results suggest the possible involvement of apoptosis signal-regulating kinase 1/c-Jun N-terminal kinase signaling in the regulation of FasL expression and subsequent apoptosis induced by resveratrol in HL-60 cells. Resveratrol also activated the small GTP-binding protein Cdc42, rather than other members such as RhoA or Rac1. Expression of a mutant Cdc42 (N17 Cdc42) dramatically reduced resveratrol-induced c-Jun N-terminal kinase activity, FasL expression and apoptotic cell death. These results showed that resveratrol induced apoptosis through the Cdc42/apoptosis signal-regulating kinase 1/c-Jun N-terminal kinase/FasL signaling cascade in HL-60 cells.